The present invention relates to methods for separating cells using immunorosettes. The invention includes novel antibody compositions for use in the method of the invention.
In many applications it is desirable to enrich or, alternatively, deplete certain cell populations in a biological sample. The fields of hematology, immunology and oncology rely on samples of peripheral blood and cell suspensions from related tissues such as bone marrow, spleen, thymus and fetal liver. The separation of specific cell types from these heterogeneous samples is key to research in these fields, diagnostics and therapy for certain malignancies and immune/hematopoietic disorders.
Purified populations of immune cells such as T cells and (antigen presenting cells are necessary for the study of immune function and are used in immunotherapy. Investigation of the cellular, molecular and biochemical processes require analysis of certain cell types in isolation. Numerous techniques have been used to isolate T cell subsets, B cells, basophils, NK cells and dendritic cells.
The isolation of hematopoietic stem cells has also been an area of great interest. Pure populations of stem cells will facilitate studies of hematopoiesis and transplantation of hematopoietic cells from peripheral blood and/or bone marrow is increasingly used in combination with high-dose chemo- and/or radiotherapy for the treatment of a variety of disorders including malignant, nonmalignant and genetic disorders. Very few cells in such transplants are capable of long-term hematopoietic reconstitution, and thus there is a strong stimulus to develop techniques for purification of hematopoietic stem cells. Furthermore, serious complications and indeed the success of a transplant procedure is to a large degree dependent on the effectiveness of the procedures that are used for the removal of cells in the transplant that pose a risk to the transplant recipient. Such cells include T lymphocytes that are responsible for graft versus host disease (GVHD) in allogenic grafts, and tumor cells in autologous transplants that may cause recurrence of the malignant growth. It is also important to debulk the graft by removing unnecessary cells and thus reducing the volume of cyropreservant to be infused.
In certain instances it is desirable to remove or deplete tumor cells from a biological sample, for example in bone marrow transplants. Epithelial cancers of the bronchi, mammary ducts and the gastrointestinal and urogenital tracts represent a major type of solid tumors seen today.
Micrometastatic tumor cell migration is thought to be an important prognostic factor for patients with epithelial cancer (Braun et al., 2000; Vaughan et al., 1990). The ability to detect such metastatic cells is limited by the effectiveness of tissue or fluid sampling and the sensitivity of tumor detection methods. A technique to enrich circulating epithelial tumor cells in blood samples would increase the ability to detect metastatic disease and facilitate the study of such rare cells and the determination of the biological changes which enable spread of disease.
Hematopoictic cells and immune cells have been separated on the basis of physical characteristics such as density and on the basis of susceptibility to certain pharmacological agents which kill cycling cells. The advent of monoclonal antibodies against cell surface antigens has greatly cxpandcd the potential to distinguish and separate distinct cell types. There are two basic approaches to separating cell populations from blood and related cell suspensions using monoclonal antibodies. They differ in whether it is the desired or undesired cells which are distinguished/labelled with the antibody(s).
In positive selection techniques the desired cells are labelled with antibodies and removed from the remaining unlabelled/unwanted cells. In negative selection, the unwanted cells are labelled and removed. Antibody/complement treatment and the use of immunotoxins are negative selection techniques, but FACS sorting and most batch wise immunoadsorption techniques can be adapted to both positive and negative selection. In immunoadsorption techniques cells are selected with monoclonal antibodies and preferentially bound to a surface which can be removed from the remainder of the cells e,g. column of beads, flasks, magnetic particles. Immunoadsorption techniques have won favour clinically and in research because they maintain the high specificity of targeting cells with monoclonal antibodies, but unlike FACSorting, they can be scaled up to deal directly with the large numbers of cells in a clinical harvest and they avoid the dangers of using cytotoxic reagents such as immunotoxins, and complement. They do however, require the use of a xe2x80x9cdevicexe2x80x9d or cell separation surface such as a column of beads, panning flask or magnet.
Current techniques for the isolation of hematopoietic stem cells, immune cells and circulating epithelial tumor cells all involve an initial step to remove red cells then antibody mediated adherence to a device or artificial particle. (Firat et al., 1988; de Wynter et al., 1975; Shpall et al., 1994; Thomas et al., 1994; Miltenyi Biotec Inc., Gladbach, Germany) In the case of positive selection there is yet another step; removal of the cells from the device or particle. All these multiple steps require time and incur cell loss. Slaper-Cortenbach et al. (1990) describes a method for purging bone marrow of common acute leukemic (cALL) cells using immunorosetting. The method requires that the erythrocytes are first removed from the bone marrow sample and are labelled with antibodies that bind to the cALL cells. The labelled erythrocytes are then added back to the sample where the cALL cells are immunorosetted. The depletion method works best when followed by an additional step of complement mediated lysis of the cALL cells.
Density Separations are commonly used to isolate peripheral blood mononuclear cells from granulocytes and erythrocytes. Ficoll (Amersham Pharmacia Biotech AB, Uppsala Sweden) is the most popular density solution used for this application. In a Ficoll density separation whole blood is layered over Ficoll, and then centrifuged. The erythrocytes and granulocytes settle to the cell pellet and the mononuclear cells remain at the Ficoll plasma interface. The success of this technique relies on the difference in density between mononuclear cells and granulocytes. If whole blood is stored for more than 24 hours the granulocytes change density and will not pellet with the red cells. Suspensions of pure mononuclear cells can not be obtained from stored blood or samples with altered cell density in a single density separation.
In view of the foregoing, there is a need in the art to provide novel methods for separating desired cells or removing unwanted cells from biological samples.
The present inventors have developed a method for separating cells by immunorosetting the cells with red blood cells or erythrocytes already existing in the sample. The method of the invention is a much simpler yet equally efficient immunoaffinity technique as compared to the prior art methods. There is no xe2x80x9cdevicexe2x80x9d or need for an artificial separation surface (eg., magnetic particles, affinity column) not normally present in the cell suspension. There is no need to first remove the erythrocytes from the sample and to then reintroduce them once they have been labelled with antibodies. Specific cell types are cross-linked to autologous erythrocytes found within the sample and subsequent rosettes are then removed by sedimentation or centrifugation.
Accordingly, in one embodiment, the present invention provides a method of separating nucleated cells from a sample comprising the nucleated cells and erythrocytes comprising:
(1) contacting the sample with an antibody composition comprising (a) at least one antibody that binds to an antigen on the nucleated cells to be separated linked, either directly or indirectly, to (b) at least one antibody that binds to the erythrocytes, under conditions to allow immunorosettes of the nucleated cells and the erythrocytes to form; and
(2) removing the immunorosettes from the sample.
The method can be used in both positive and negative selection protocols. The method can be used on any sample that contains red blood cells including whole blood, bone marrow, fetal liver, cord blood, buffy coat suspensions, pleural and peritoneal effusion and, samples of thymocytes and splenocytes.